Photolabile nucleoside protecting groups

ABSTRACT

A synthetic strategy for the creation of large scale chemical diversity. Solid-phase chemistry, photolabile protecting groups, and photolithography are used to achieve light-directed spatially-addressable parallel chemical synthesis. Binary masking techniques are utilized in one embodiment. A reactor system, photoremovable protective groups, and improved data collection and handling techniques are also disclosed. A technique for screening linker molecules is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of Ser. No. 07/624,120, filed Dec. 6,1990, now abandoned which is a application is a continuation-in-part ofSer. No. 07/492,462, now application U.S. Pat. No. 5,143.859 filed Mar.7, 1990, which is a continuation-in-part of application Ser. No.362,901, filed Jun. 7, 1989 now abandoned, assigned to the assignee ofthe present application, and incorporated herein by reference for allpurposes. This application is related to applications with Ser. Nos.07/626,730 now U.S. Pat. No. 5,547,839 and 07/624,114 now abandonedfiled on the same day as the present application, and also incorporatedherein by reference for all purposes.

COPYRIGHT NOTICE Microfiche Appendix

This specification includes microfiche Appendices A and B, having 3sheets with 226 frames.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

BACKGROUND OF THE INVENTION

The present invention relates to the field of polymer synthesis. Morespecifically, the invention provides a reactor system, a maskingstrategy, photoremovable protective groups, data collection andprocessing techniques, and applications for light directed synthesis ofdiverse polymer sequences on substrates.

SUMMARY OF THE INVENTION

Methods, apparatus, and compositions for synthesis and use of diversepolymer sequences on a substrate are disclosed, as well as applicationsthereof.

According to one aspect of the invention, an improved reactor system forsynthesis of diverse polymer sequences on a substrate is providedAccording to this embodiment the invention provides for a reactor forcontacting reaction fluids to a substrate; a system for deliveringselected reaction fluids to the reactor; a translation stage for movinga mask or substrate from at least a first relative location relative toa second relative location; a light for illuminating the substratethrough a mask at selected times; and an appropriately programmeddigital computer for selectively directing a flow of fluids from thereactor system, selectively activating the translation stage, andselectively illuminating the substrate so as to form a plurality ofdiverse polymer sequences on the substrate at predetermined locations.

The invention also provides a technique for selection of linkermolecules in a very large scale immobilized polymer synthesis (VLSIPS™)method. According to this aspect of the invention, the inventionprovides a method of screening a plurality of linker polymers for use inbinding affinity studies. The invention includes the steps of forming aplurality of linker polymers on a substrate in selected regions, thelinker polymers formed by the steps of recursively: on a surface of asubstrate, irradiating a portion of the selected regions to remove aprotective group, and contacting the surface with a monomer; contactingthe plurality of linker polymers with a ligand; and contacting theligand with a labeled receptor.

According to another aspect of the invention, improved photoremovableprotective groups are provided. According to this aspect of theinvention a compound having the formula: ##STR1## wherein n=0 or 1; Y isselected from the group consisting of an oxygen of the carboxyl group ofa natural or unnatural amino acid, an amino group of a natural orunnatural amino acid, or the C-5' oxygen group of a natural or unnaturaldeoxyribonucleic or ribonucleic acid; R¹ and R² independently are ahydrogen atom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl,thiol, thioether, amino, nitro, carboxyl, formate, formamido, sulfido,or phosphido group; and R³ is; a alkoxy, alkyl, aryl, hydrogen, oralkenyl group is provided.

The invention also provides improved masking techniques for the VLSIPS™methodology. According to one aspect of the masking technique, theinvention provides an ordered method for forming a plurality of polymersequences by sequential addition of reagents comprising the step ofserially protecting and deprotecting portions of the plurality ofpolymer sequences for addition of other portions of the polymersequences using a binary synthesis strategy.

Improved data collection equipment and techniques are also provided.According to one embodiment, the instrumentation provides a system fordetermining affinity of a receptor to a ligand comprising: means forapplying light to a surface of a substrate, the substrate comprising aplurality of ligands at predetermined locations, the means for providingsimultaneous illumination at a plurality of the predetermined locations;and an array of detectors for detecting light fluoresced at theplurality of predetermined locations. The invention further provides forimproved data analysis techniques including the steps of exposingfluorescently labelled receptors to a substrate, the substratecomprising a plurality of ligands in regions at known locations; at aplurality of data collection points within each of the regions,determining an amount of light fluoresced from the data collectionpoints; removing the data collection points deviating from apredetermined statistical distribution; and determining a relativebinding affinity of the receptor to remaining data collection points.

Protected amino acid N-carboxy anhydrides for use in polymer synthesisare also disclosed. According to this aspect the invention provides acompound having the formula: ##STR2## where R is a side chain of anatural or unnatural amino acid and X is a photoremovable protectinggroup.

A further understanding of the nature and advantages of the inventionsherein may be realized by reference to the remaining portions of thespecification and the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates light-directed spatially-addressableparallel chemical synthesis;

FIG. 2 schematically illustrates one example of light-directed peptidesynthesis;

FIG. 3 is a three-dimensional representation of a portion of thecheckerboard array of YGGFL and PGGFL;

FIG. 4 schematically illustrates the automated system for synthesizingdiverse polymer sequences;

FIG. 5a and 5b illustrate operation of a program for polymer sythesis;

FIG. 6 is a schematic illustration of a "pure" binary masking strategy;

FIG. 7 is a schematic illustration of a gray code binary maskingstrategy;

FIG. 8 is a schematic illustration of a modified gray code binarymasking strategy;

FIG. 9a schematically illustrates a masking scheme for a four stepsynthesis;

FIG. 9b schematically illustrates synthesis of all 400 peptide dimers;

FIG. 10 is a coordinate map for the ten-step binary synthesis;

FIG. 11 schematically illustrates a data collection system;

FIG. 12 is a block diagram illustrating the architecture of the datacollection system;

FIG. 13 is a flow chart illustrating operation of software for the datacollection/analysis system; and

FIG. 14 illustrates a three-dimensional plot of intensity versusposition for light directed synthesis of a dinucleotide.

DESCRIPTION OF THE PREFERRED EMBODIMENTS CONTENTS

I. Definitions

II. General

A. Deprotection and Addition

1. Example

2. Example

B. Antibody recognition

1. Example

III. Synthesis

A. Reactor System

B. Binary Synthesis Strategy

1. Example

2. Example

3. Example

4. Example

5. Example

6. Example

C. Linker Selection

D. Protecting Groups

1. Use of Photoremovable Groups During Solid-Phase Synthesis of Peptides

2. Use of Photoremovable Groups During Solid-Phase Synthesis ofOligonucleotides

E. Amino Acid N-Carboxy Anhydrides Protected with a Photoremovable Group

IV. Data Collection

A. Data Collection System

B. Data Analysis

V. Other Representative Applications

A. Oligonucleotide Synthesis

1. Example

VI. Conclusion

I. Definitions

Certain terms used herein are intended to have the following generaldefinitions:

1. Complementary: Refers to the topological compatibility or matchingtogether of interacting surfaces of a ligand molecule and its receptor.Thus, the receptor and its ligand can be described as complementary, andfurthermore, the contact surface characteristics are complementary toeach other.

2. Epitope: The portion of an antigen molecule which is delineated bythe area of interaction with the subclass of receptors known asantibodies.

3. Ligand: A ligand is a molecule that is recognized by a particularreceptor. Examples of ligands that can be investigated by this inventioninclude, but are not restricted to, agonists and antagonists for cellmembrane receptors, toxins and venoms, viral epitopes, hormones, hormonereceptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g.opiates, steroids, etc.), lectins, sugars, oligonucleotides, nucleicacids, oligosaccharides, proteins, and monoclonal antibodies.

4. Monomer: A member of the set of small molecules which can be joinedtogether to form a polymer. The set of monomers includes but is notrestricted to, for example, the set of common L-amino acids, the set ofD-amino acids, the set of synthetic amino acids, the set of nucleotidesand the set of pentoses and hexoses. As used herein, monomers refers toany member of a basis set for synthesis of a polymer. For example,dimers of the 20 naturally occurring L-amino acids form a basis set of400 monomers for synthesis of polypeptides. Different basis sets ofmonomers may be used at successive steps in the synthesis of a polymer.Furthermore, each of the sets may include protected members which aremodified after synthesis.

5. Peptide: A polymer in which the monomers are alpha amino acids andwhich are joined together through amide bonds and alternatively referredto as a polypeptide. In the context of this specification it should beappreciated that the amino acids may be the L-optical isomer or theD-optical isomer. Peptides are often two or more amino acid monomerslong, and often more than 20 amino acid monomers long. Standardabbreviations for amino acids are used (e.g., P for proline). Theseabbreviations are included in Stryer, Biochemistry, Third Ed., 1988,which is incorporated herein by reference for all purposes.

6. Radiation: Energy which may be selectively applied including energyhaving a wavelength of between 10⁻¹⁴ and 10⁴ meters including, forexample, electron beam radiation, gamma radiation, x-ray radiation,ultraviolet radiation, visible light, infrared radiation, microwaveradiation, and radio waves. "Irradiation" refers to the application ofradiation to a surface.

7. Receptor: A molecule that has an affinity for a given ligand.Receptors may be naturally-occurring or manmade molecules. Also, theycan be employed in their unaltered state or as aggregates with otherspecies. Receptors may be attached, covalently or noncovalently, to abinding member, either directly or via a specific binding substance.Examples of receptors which can be employed by this invention include,but are not restricted to, antibodies, cell membrane receptors,monoclonal antibodies and antisera reactive with specific antigenicdeterminants (such as on viruses, cells or other materials), drugs,polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars,polysaccharides, cells, cellular, membranes, and organelles. Receptorsare sometimes referred to in the art as anti-ligands. As the termreceptors is used herein, no difference in meaning is intended. A"Ligand Receptor Pair" is formed when two macromolecules have combinedthrough molecular recognition to form a complex.

Other examples of receptors which can be investigated by this inventioninclude but are not restricted to:

a) Microorganism receptors: Determination of ligands which bind toreceptors, such as specific transport proteins or enzymes essential tosurvival of microorganisms, is useful in developing a new class ofantibiotics. Of particular value would be antibiotics againstopportunistic fungi, protozoa, and those bacteria resistant to theantibiotics in current use.

b) Enzymes: For instance, one type of receptor is the binding site ofenzymes such as the enzymes responsible for cleaving neurotransmitters;determination of ligands which bind to certain receptors to modulate theaction of the enzymes which cleave the different neurotransmitters isuseful in the development of drugs which can be used in the treatment ofdisorders of neurotransmission.

c) Antibodies: For instance, the invention may be useful ininvestigating the ligand-binding site on the antibody molecule whichcombines with the epitope of an antigen of interest; determining asequence that mimics an antigenic epitope may lead to the development ofvaccines of which the immunogen is based on one or more of suchsequences or lead to the development of related diagnostic agents orcompounds useful in therapeutic treatments such as for autoimmunediseases (e.g., by blocking the binding of the "self" antibodies).

d) Nucleic Acids: Sequences of nucleic acids may be synthesized toestablish DNA or RNA binding sequences.

e) Catalytic Polypeptides: Polymers, preferably polypeptides, which arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products. Such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,which functionality is capable of chemically modifying the boundreactant. Catalytic polypeptides are described in, for example,application U.S. Pat. No. 5,215,899, which is incorporated herein byreference for all purposes.

f) Hormone receptors: Example of hormone receptors include, e.g. thereceptors for insulin and growth hormone. Determination of the ligandswhich bind with high affinity to a receptor is useful in the developmentof, for example, an oral replacement of the daily injections whichdiabetics must take to relieve the symptoms of diabetes, and in theother case, a replacement for the scarce human growth hormone which canonly be obtained from cadavers or by recombinant DNA technology. Otherexamples are the vasoconstrictive hormone receptors; determination ofthose ligands which bind to a receptor may lead to the development ofdrugs to control blood pressure.

g) Opiate receptors: Determination of ligands which bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

8. Substrate: A material having a rigid or semi-rigid surface. In manyembodiments, at least one surface of the substrate will be substantiallyflat, although in some embodiments it may be desirable to physicallyseparate synthesis regions for different polymers with, for example,wells, raised regions, etched trenches, or the like. According to otherembodiments, small beads may be provided on the surface which may bereleased upon completion of the synthesis.

9. Protective Group: A material which is chemically bound to a monomerunit and which may be removed upon selective exposure to an activatorsuch as electromagnetic radiation. Examples of protective groups withutility herein include those comprising nitropiperonyl,pyrenylmethoxy-carbonyl, nitroveratryl, nitrobenzyl, dimethyldimethoxybenzyl, 5-bromo-7-nitroindolinyl, o-hydroxy-α-methyl cinnamoyl,and 2-oxymethylene anthraquinone.

10. Predefined Region: A predefined region is a localized area on asurface which is, was, or is intended to be activated for formation of apolymer. The predefined region may have any convenient shape, e.g.,circular, rectangular, elliptical, wedgeshaped, etc. For the sake ofbrevity herein, "predefined regions" are sometimes referred to simply as"regions."

11. Substantially Pure: A polymer is considered to be "substantiallypure" within a predefined region of a substrate when it exhibitscharacteristics that distinguish it from other predefined regions.Typically, purity will be measured in terms of biological activity orfunction as a result of uniform sequence. Such characteristics willtypically be measured by way of binding with a selected ligand orreceptor.

12. Activator refers to an energy source adapted to render a groupactive and which is directed from a source to a predefined location on asubstrate. A primary illustration of an activator is light. Otherexamples of activators include ion beams, electric fields, magneticfields, electron beams, x-ray, and the like.

13. Binary Synthesis Strategy refers to an ordered strategy for parallelsynthesis of diverse polymer sequences by sequential addition ofreagents which may be represented by a reactant matrix, and a switchmatrix, the product of which is a product matrix. A reactant matrix is a1×n matrix of the building blocks to be added. The elements of theswitch matrix are binary numbers. In preferred embodiments, a binarystrategy is one in which at least two successive steps illuminate halfof a region of interest on the substrate. In most preferred embodiments,binary synthesis refers to a synthesis strategy which also factors aprevious addition step. For example, a strategy in which a switch matrixfor a masking strategy halves regions that were previously illuminated,illuminating about half of the previously illuminated region andprotecting the remaining half (while also protecting about half ofpreviously protected regions and illuminating about half of previouslyprotected regions). It will be recognized that binary rounds may beinterspersed with non-binary rounds and that only a portion of asubstrate may be subjected to a binary scheme, but will still beconsidered to be a binary masking scheme within the definition herein. Abinary "masking" strategy is a binary synthesis which uses light toremove protective groups from materials for addition of other materialssuch as amino acids. In preferred embodiments, selected columns of theswitch matrix are arranged in order of increasing binary numbers in thecolumns of the switch matrix.

14. Linker refers to a molecule or group of molecules attached to asubstrate and spacing a synthesized polymer from the substrate forexposure/binding to a receptor.

II. General

The present invention provides synthetic strategies and devices for thecreation of large scale chemical diversity. Solid-phase chemistry,photolabile protecting groups, and photolithography are brought togetherto achieve light-directed spatially-addressable parallel chemicalsynthesis in preferred embodiments.

The invention is described herein for purposes of illustration primarilywith regard to the preparation of peptides and nucleotides, but couldreadily be applied in the preparation of other polymers. Such polymersinclude, for example, both linear and cyclic polymers of nucleic acids,polysaccharides, phospholipids, and peptides having either α-, β-, orω-amino acids, heteropolymers in which a known drug is covalently boundto any of the above, polyurethanes, polyesters, polycarbonates,polyureas, polyamides, polyethyleneimines, polyarylene sulfides,polysiloxanes, polyimides, polyacetates, or other polymers which will beapparent upon review of this disclosure. It will be recognized further,that illustrations herein are primarily with reference to C- toN-terminal synthesis, but the invention could readily be applied to N-to C-terminal synthesis without departing from the scope of theinvention.

A. Deprotection and Addition

The present invention uses a masked light source or other activator todirect the simultaneous synthesis of many different chemical compounds.FIG. 1 is a flow chart illustrating the process of forming chemicalcompounds according to one embodiment of the invention. Synthesis occurson a solid support 2. A pattern of illumination through a mask 4a usinga light source 6 determines which regions of the support are activatedfor chemical coupling. In one preferred embodiment activation isaccomplished by using light to remove photolabile protecting groups fromselected areas of the substrate.

After deprotection, a first of a set of building blocks (indicated by"A" in FIG. 1), each bearing a photolabile protecting group (indicatedby "X") is exposed to the surface of the substrate and it reacts withregions that were addressed by light in the preceding step. Thesubstrate is then illuminated through a second mask 4b, which activatesanother region for reaction with a second protected building block "B".The pattern of masks used in these illuminations and the sequence ofreactants define the ultimate products and their locations, resulting indiverse sequences at predefined locations, as shown with the sequencesACEG and BDFH in the lower portion of FIG. 1. Preferred embodiments ofthe invention take advantage of combinatorial masking strategies to forma large number of compounds in a small number of chemical steps.

A high degree of miniaturization is possible because the density ofcompounds is determined largely with regard to spatial addressability ofthe activator, in one case the diffraction of light. Each compound isphysically accessible and its position is precisely known. Hence, thearray is spatially-addressable and its interactions with other moleculescan be assessed.

In a particular embodiment shown in FIG. 1, the substrate contains aminogroups that are blocked with a photolabile protecting group. Amino acidsequences are made accessible for coupling to a receptor by removal ofthe photoprotective groups.

When a polymer sequence to be synthesized is, for example, apolypeptide, amino groups at the ends of linkers attached to a glasssubstrate are derivatized with nitroveratryloxycarbonyl (NVOC), aphotoremovable protecting group. The linker molecules may be, forexample, aryl acetylene, ethylene glycol oligomers containing from 2-10monomers, diamines, diacids, amino acids, or combinations thereof.Photiodeprotection is effected by illumination of the substrate through,for example, a mask wherein the pattern has transparent regions withdimensions of, for example, less than 1 cm², 10⁻¹ cm², 10⁻² cm ², 10⁻³cm ², 10⁻⁴ cm ², 10⁻⁵ cm ², 10⁻⁶ cm², 10⁻⁷ cm ², 10⁻⁸ cm², or 10⁻¹⁰ cm². In a preferred embodiment, the regions are between about 10×10 μm and500×500 μm. According to some embodiments, the masks are arranged toproduce a checkerboard array of polymers, although any one of a varietyof geometric configurations may be utilized.

1. Example

In one example of the invention, free amino groups were fluorescentlylabelled by treatment of the entire substrate surface with fluoresceinisothiocynate (FITC) after photodeprotection. Glass microscope slideswere cleaned, aminated by treatment with 0.1% aminopropyltriethoxysilanein 95% ethanol, and incubated at 110° C. for 20 min. The aminatedsurface of the slide was then exposed to a 30 mM solution of theN-hydroxysuccinimide ester of NVOC-GABA(nitroveratryloxycarbonyl-τ-amino butyric acid) in DMF. The NVOCprotecting group was photolytically removed by imaging the 365 nm outputfrom a Hg arc lamp through a chrome on glass 100 μm checkerboard maskonto the substrate for 20 min at a power density of 12 mW/cm². Theexposed surface was then treated with 1 mM FITC in DMF. The substratesurface was scanned in an epi-fluorescence microscope (Zeiss Axioskop20) using 488 nm excitation from an argon ion laser (Spectra-Physicsmodel 2025). The fluorescence emission above 520 nm was detected by acooled photomultiplier (Hamamatsu 943-02) operated in a photon countingmode. Fluorescence intensity was translated into a color display withred in the highest intensity and black in the lowest intensity areas.The presence of a high-contrast fluorescent checkerboard pattern of100×100 μm elements revealed that free amino groups were generated inspecific regions by spatially-localized photodeprotection.

2. Example

FIG. 2 is a flow chart illustrating another example of the invention.Carboxy-activated NVOC-leucine was allowed to react with an aminatedsubstrate. The carboxy activated HOBT ester of leucine and other aminoacids used in this synthesis was formed by mixing 0.25 mmol of the NVOCamino protected amino acid with 37 mg HOBT (l-hydroxybenzotriazole), 111mg BOP (benzotriazolyl-n-oxy-tris(dimethylamino)-phosphoniumhexa-fluorophosphate) and 86 μl DIEA(diisopropylethylamine) in 2.5 ml DMF. The NVOC protecting group wasremoved by uniform illumination. Carboxy-activated NVOC-phenylalaninewas coupled to the exposed amino groups for 2 hours at room temperature,and then washed with DMF and methylene chloride. Two unmasked cycles ofphotodeprotection and coupling with carboxy-activated NVOC-glycine werecarried out. The surface was then illuminated through a chrome on glass50 μm checkerboard pattern mask. Carboxy-activatedNα-tBOC-O-tButyl-L-tyrosine was then added. The entire surface wasuniformly illuminated to photolyze the remaining NVOC groups. Finally,carboxy-activated NVOC-L-proline was added, the NVOC group was removedby illumination, and the t-BOC and t-butyl protecting groups wereremoved with TFA. After removal of the protecting groups, the surfaceconsisted of a 50 μm checkerboard array of Tyr-Gly-Gly-Phe-Leu (YGGFL)(SEQ ID NO:1)and Pro-Gly-Gly-Phe-Leu (PGGFL) (SEQ ID NO:2).

B. Antibody Recognition

In one preferred embodiment the substrate is used to determine which ofa plurality of amino acid sequences is recognized by an antibody ofinterest.

1. Example

In one example, the array of pentapeptides in the example illustrated inFIG. 2 was, probed with a mouse monoclonal antibody directed againstβ-endorphin. This antibody (called 3E7) is known to bind YGGFL and YGGFM(SEQ ID NO:21) with nanomolar affinity and is discussed in Meo et al.,Proc. Natl. Acad. Sci. USA (1983) 80:4084, which is incorporated byreference herein for all purposes. This antibody requires the aminoterminal tyrosine for high affinity binding. The array of peptidesformed as described in FIG. 2 was incubated with a 2 μg/ml mousemonoclonal antibody (3E7) known to recognize YGGFL. 3E7 does not bindPGGFL. A second incubation with fluoresceinated goat anti-mouse antibodylabeled the regions that bound 3E7. The surface was scanned with anepi-fluorescence microscope. The results showed alternating bright anddark 50 μm squares indicating that YGGFL and PGGFL were synthesized ingeometric array determined by the mask. A high contrast (>12:1 intensityratio) fluorescence checkerboard image shows that (a) YGGFL and PGGFLwere synthesized in alternate 50 μm squares, (b) YGGFL attached to thesurface is accessible for binding to antibody 3E7, and (c) antibody 3E7does not bind to PGGFL.

A three-dimensional representation of the fluorescence intensity data ina portion of the checkboard is shown in FIG. 3. This figure shows thatthe border between synthesis sites is sharp. The height of each spike inthis display is linearly proportional to the integrated fluorescenceintensity in a 2.5 μm pixel. The transition between PGGFL and YGGFLoccurs within two spikes (5 μm). There is little variation in thefluorescence intensity of different YGGFL squares. The mean intensity ofsixteen YGGFL synthesis sites was 2.03×10⁵ counts and the standarddeviation was 9.6×10³ counts.

III. Synthesis

A. Reactor System

FIG. 4 schematically illustrates a device used to synthesize diversepolymer sequences on a substrate. The device includes an automatedpeptide synthesizer 401. The automated peptide synthesizer is a devicewhich flows selected reagents through a flow cell 402 under thedirection of a computer 404. In a preferred embodiment the synthesizeris an ABI Peptide Synthesizer, model no. 431A. The computer may beselected from a wide variety of computers or discrete logic includingfor, example, an IBM PC-AT or similar computer linked with appropriateinternal control systems in the peptide synthesizer. The PC is providedwith signals from the board computer indicative of, for example, the endof a coupling cycle.

Substrate 406 is mounted on the flow cell, forming a cavity between thesubstrate and the flow cell. Selected reagents flow through this cavityfrom the peptide synthesizer at selected times, forming an array ofpeptides on the face of the substrate in the cavity. Mounted above thesubstrate, and preferably in contact with the substrate is a mask 408.Mask 408 is transparent in selected regions to a selected wavelength oflight and is opaque in other regions to the selected wavelength oflight. The mask is illuminated with a light source 410 such as a UVlight source. In one specific embodiment the light source 410 is a modelno. 82420 made by Oriel. The mask is held and translated by an x-y-ztranslation stage 412 such as an x-y translation stage made by NewportCorp. The computer coordinates action of the peptide synthesizer, x-ytranslation stage, and light source. Of course, the invention may beused in some embodiments with translation of the substrate instead ofthe mask.

In operation, the substrate is mounted on the reactor cavity. The slide,with its surface protected by a suitable photo removable protectivegroup, is exposed to light at selected locations by positioning the maskand illuminating the light source for a desired period of time (such as,for example, 1 sec to 60 min in the case of peptide synthesis). Aselected peptide or other monomer/polymer is pumped through the reactorcavity by the peptide synthesizer for binding at the selected locationson the substrate. After a selected reaction time (such as about 1 sec to300 min in the case of peptide reactions) the monomer is washed from thesystem, the mask is appropriately repositioned or replaced, and thecycle is repeated. In most embodiments of the invention, reactions maybe conducted at or near ambient temperature.

FIGS. 5a and 5b are flow charts of the software used in operation of thereactor system. At step 502 the peptide synthesis software isinitialized. At step 504 the system calibrates positioners on the x-ytranslation stage and begins a main loop. At step 506 the systemdetermines which, if any, of the function keys on the computer have beenpressed. If F1 has been pressed, the system prompts the user for inputof a desired synthesis process. If the user enters F2, the system allowsa user to edit a file for a synthesis process at step 510. If the userenters F3 the system loads a process from a disk at step 512. If theuser enters F4 the system saves an entered or edited process to disk atstep 514. If the user selects F5 the current process is displayed atstep 516 while selection of F6 starts the main portion of the program,i.e., the actual synthesis according to the selected process. If theuser selects F7 the system displays the location of the synthesizedpeptides, while pressing F10 returns the user to the disk operatingsystem.

FIG. 5b illustrates the synthesis step 518 in greater detail. The mainloop of the program is started in which the system first moves the maskto a next position at step 526. During the main loop of the program,necessary chemicals flow through the reaction cell under the directionof the on-board computer in the peptide synthesizer. At step 528 thesystem then waits for an exposure command and, upon receipt of theexposure command exposes the substrate for a desired time at step 530.When an acknowledge of exposure complete is received at step 532 thesystem determines if the process is complete at step 534 and, if so,waits for additional keyboard input at step 536 and, thereafter, exitsthe perform synthesis process.

A computer program used for operation of the system described above isincluded as microfiche Appendix A (Copyright, 1990, Affymax TechnologiesN.V., all rights reserved). The program is written in Turbo C++ (BorlandInt'l) and has been implemented in an IBM compatible system. The motorcontrol software is adapted from software produced by NewportCorporation. It will be recognized that a large variety of programminglanguages could be utilized without departing from the scope of theinvention herein. Certain calls are made to a graphics program in"Programmer Guide to PC and PS2 Video Systems" (Wilton, Microsoft Press,1987), which is incorporated herein by reference for all purposes.

Alignment of the mask is achieved by one of two methods in preferredembodiments. In a first embodiment the system relies upon relativealignment of the various components, which is normally acceptable sincex-y-z translation stages are capable of sufficient accuracy for thepurposes herein. In alternative Embodiments, alignment marks on thesubstrate are coupled to a CCD device for appropriate alignment.

According to some embodiments, pure reagents are not added at each step,or complete photolysis of the protective groups is not provided at eachstep. According to these embodiments, multiple products will be formedin each synthesis site. For example, if the monomers A and B are mixedduring a synthesis step, A and B will bind to deprotected regions,roughly in proportion to their concentration in solution. Hence, amixture of compounds will be formed in a synthesis region. A substrateformed with mixtures of compounds in various synthesis regions may beused to perform, for example, an initial screening of a large number ofcompounds, after which a smaller number of compounds in regions whichexhibit high binding affinity are further screened. Similar results maybe obtained by only partially photylizing a region, adding a firstmonomer, re-photylizing the same region, and exposing the region to asecond monomer.

B. Binary Synthesis Strategy

In a light-directed chemical synthesis, the products formed depend onthe pattern and order of masks, and on the order of reactants. To make aset of products there will in general be "n" possible masking schemes.In preferred embodiments of the invention herein a binary synthesisstrategy is utilized. The binary synthesis strategy is illustratedherein primarily with regard to a masking strategy, although it will beapplicable to other polymer synthesis strategies such as the pinstrategy, and the like.

In a binary synthesis strategy, the substrate is irradiated with a firstmask, exposed to a first building block, irradiated with a second mask,exposed to a second building block, etc. Each combination of maskedirradiation and exposure to a building block is referred to herein as a"cycle."

In a preferred binary masking scheme, the masks for each cycle allowirradiation of half of a region of interest on the substrate andprotection of the remaining half of the region of interest. By "half" itis intended herein not to mean exactly one-half the region of interest,but instead a large fraction of the region of interest such as fromabout 30 to 70 percent of the region of interest. It will be understoodthat the entire masking scheme need not take a binary form; insteadnon-binary cycles may be introduced as desired between binary cycles.

In preferred embodiments of the binary masking scheme, a given cycleilluminates only about half of the region which was illuminated in aprevious cycle, while protecting the remaining half of the illuminatedportion from the previous cycle. Conversely, in such preferredembodiments, a given cycle illuminates half of the region which wasprotected in the previous cycle and protects half the region which wasprotected in a previous cycle.

The synthesis strategy is most readily illustrated and handled in matrixnotation. At each synthesis site, the determination of whether to add agiven monomer is a binary process. Therefore, each product element P_(j)is given by the dot product of two vectors, a chemical reactant vector,e.g., C= A,B,C,D!, and a binary vector σ_(j). Inspection of the productsin the example below for a four-step synthesis, shows that in onefour-step synthesis σ₁ = 1,0,1,0!, σ₂ = 1,0,0,1!, σ₃ = 0,1,1,0!, and σ₄= 0,1,0,1!, where a 1 indicates illumination and a 0 indicatesprotection. Therefore, it becomes possible to build a "switch matrix" Sfrom the column vectors σ_(j) (j=l,k where k is the number of products).##EQU1## The outcome P of a synthesis is simply P=CS, the product of thechemical reactant matrix and the switch matrix.

The switch matrix for an n-cycle synthesis yielding k products has nrows and k columns. An important attribute of S is that each rowspecifies a mask. A two-dimensional mask m_(j) for the jth chemical stepof a synthesis is obtained directly from the jth row of S by placing theelements s_(j1), . . . s_(jk) into, for example, a square format. Theparticular arrangement below provides a square format, although linearor other arrangements may be utilized. ##EQU2##

Of course, compounds formed in a light-activated synthesis can bepositioned in any defined geometric array. A square or rectangularmatrix is convenient but not required. The rows of the switch matrix maybe transformed into any convenient array as long as equivalenttransformations are used for each row.

For example, the masks in the four-step synthesis below are then denotedby: ##EQU3## where 1 denotes illumination (activation) and 0 denotes noillumination.

The matrix representation is used to generate a desired set of productsand product maps in preferred embodiments. Each compound is defined bythe product of the chemical vector and a particular switch vector.Therefore, for each synthesis address, one simply saves the switchvector, assembles all of them into a switch matrix, and extracts each ofthe rows to form the masks.

In some cases, particular product distributions or a maximal number ofproducts are desired. For example, for C= A,B,C,D!, any switch vector(σ_(j)) consists of four bits. Sixteen four-bit vectors exist. Hence, amaximum of 16 different products can be made by sequential addition ofthe reagents A,B,C,D!. These 16 column vectors can be assembled in 16|different ways to form a switch matrix. The order of the column vectorsdefines the masking patterns, and therefore, the spatial ordering ofproducts but not their makeup. One ordering of these columns gives thefollowing switch matrix (in which "null" (φ) additions are included inbrackets for the sake of completeness, although such null additions areelsewhere ignored herein): ##EQU4## The columns of S according to thisaspect of the invention are the binary representations of the numbers 15to 0. The sixteen products of this binary synthesis are ABCD, ABC, ABD,AB, ACD, AC, AD, A, BCD, BC, BD, B, CD, C, D, and φ (null). Also notethat each of the switch vectors from the four-step synthesis masks above(and hence the synthesis products) are present in the four bit binaryswitch matrix. (See columns 6, 7, 10, and 11)

This synthesis procedure provides an easy way for mapping the completedproducts. The products in the various locations on the substrate aresimply defined by the columns of the switch matrix (the first columnindicating, for example, that the product ABCD will be present in theupper left-hand location of the substrate). Furthermore, if onlyselected desired products are to be made, the mask sequence can bederived by extracting the columns with the desired sequences. Forexample, to form the product set ABCD, ABD, ACD, AD, BCD, BD, CD, and D,the masks are formed by use of a switch matrix with only the 1st, 3rd,5th, 7th, 9th, 11th, 13th, and 15th columns arranged into the switchmatrix: ##EQU5## To form all of the polymers of length 4, the reactantmatrix ABCDABCDABCDABCD! is used. The switch matrix will be formed froma matrix of the binary numbers from 0 to 2¹⁶ arranged in columns. Thecolumns having four monomers are than selected and arranged into aswitch matrix. Therefore, it is seen that the binary switch matrix ingeneral will provide a representation of all the products which can bemade from an n-step synthesis, from which the desired products are thenextracted.

The rows of the binary switch matrix will, in preferred embodiments,have the property that each masking step illuminates half of thesynthesis area. Each masking step also factors the preceding maskingstep; that is, half of the region that was illuminated in the precedingstep is again illuminated, whereas the other half is not. Half of theregion that was unilluminated in the preceding step is also illuminated,whereas the other half is not. Thus, masking is recursive. The masks areconstructed, as described previously, by extracting the elements of eachrow and placing them in a square array. For example, the four masks in Sfor a four-step synthesis are: ##EQU6##

The recursive factoring of masks allows the products of a light-directedsynthesis to be represented by a polynomial. (Some light activatedsyntheses can only be denoted by irreducible, i.e., prime polynomials.)For example, the polynomial corresponding to the top synthesis of FIG.9a (discussed below) is

    P=(A+B)(C+D)

A reaction polynomial may be expanded as though it were an algebraicexpression, provided that the order of joining of reactants X₁ and X₂ ispreserved (X₁ X₂ ≠X₂ X₁), i.e., the products are not commutative. Theproduct then is AC+AD+BC+BD. The polynomial explicitly specifies thereactants and implicitly specifies the mask for each step. Each pair ofparentheses demarcates a round of synthesis. The chemical reactants of around (e.g., A and B) react at nonoverlapping sites and hence cannotcombine with one other. The synthesis area is divided equally amongstthe elements of a round (e.g., A is directed to one-half of the area andB to the other half). Hence, the masks for a round (e.g., the masksm_(A) and m_(B)) are orthogonal and form an orthonormal set. Thepolynomial notation also signifies that each element in a round is to bejoined to each element of the next round (e.g., A with C, A with D, Bwith C, and B with D). This is accomplished by having m_(C) overlapm_(A) an m_(B) equally, and likewise for m_(D). Because C and D areelements of a round, M_(C) and m_(D) are orthogonal to each other andform an orthonormal set.

The polynomial representation of the binary synthesis described above,in which 16 products are made from 4 reactants, is

    P=(A+φ) (B+φ) (C+φ) (D+φ)

which gives ABCD, ABC, ABD, AB, ACD, AC, AD, A, BCD, BC, BD, B, CD, C,D, and φ when expanded (with the rule that φX=X and Xφ=X, andremembering that joining is ordered). In a binary synthesis, each roundcontains one reactant and one null (denoted by φ). Half of the synthesisarea receives the reactant and the other half receives nothing. Eachmask overlaps every other mask equally.

Binary rounds and non-binary rounds can be interspersed as desired, asin

    P=(A+φ) (B) (C+D+φ) (E+F+G)

The 18 compounds formed are ABCE, ABCF, ABCG, ABDE, ABDF, ABDG, ABE,ABF, ABG, BCE, BCF, BCG, BDE, BDF, BDG, BE, BF, and BG. The switchmatrix S for this 7-step synthesis is ##EQU7## The round denoted by (B)places B in all products because the reaction area was uniformlyactivated (the mask for B consisted entirely of 1's).

The number of compounds k formed in a synthesis consisting of r rounds,in which the ith round has b_(i) chemical reactants and z_(i) nulls, is

    k=Σ(b.sub.i +z.sub.i)

and the number of chemical steps n is

    n=Σb.sub.i

The number of compounds synthesized when b=a and z=0 in all rounds isa^(n/a), compared with 2^(n) for a binary synthesis. For n=20 and a=5,625 compounds (all tetramers) would be formed, compared with 1.049×10⁶compounds in a binary synthesis with the same number of chemical steps.

It should also be noted that rounds in a polynomial can be nested, as in

    (A+(B+φ)(C+φ)) (D+φ)

The products are AD, BCD, BD, CD, D, A, BC, B, C, and φ.

Binary syntheses are attractive for two reasons. First, they generatethe maximal number of products (2^(n)) for a given number of chemicalsteps (n). For four reactants, 16 compounds are formed in the binarysynthesis, whereas only 4 are made when each round has two reactants. A10-step binary synthesis yields 1,024 compounds, and a 20-step synthesisyields 1,048,576. Second, products formed in a binary synthesis are acomplete nested set with lengths ranging from 0 to n. All compounds thatcan be formed by deleting one or more units from the longest product(the n-mer) are present. Contained within the binary set are the smallersets that would be formed from the same reactants using any other set ofmasks (e.g., AC, AD, BC, and BD formed in the synthesis shown in FIG. 6are present in the set of 16 formed by the binary synthesis). In somecases, however, the experimentally achievable spatial resolution may notsuffice to accommodate all the compounds formed. Therefore, practicallimitations may require one to select a particular subset of thepossible switch vectors for a given synthesis.

1. Example

FIG. 6 illustrates a synthesis with binary masking scheme. The binarymasking scheme provides the greatest number of sequences for a givennumber of cycles. According to this embodiment, a mask ml allowsillumination of half of the substrate. The substrate is then exposed tothe building block A, which binds at the illuminated regions.

Thereafter, the mask m2 allows illumination of half of the previouslyilluminated region, while protecting half of the previously illuminatedregion. The building block B is then added, which binds at theilluminated regions from m2.

The process continues with masks m3, m4, and m5, resulting in theproduct array shown in the bottom portion of the figure. The processgenerates 32 (2 raised to the power of the number of monomers) sequenceswith 5 (the number of monomers) cycles.

2. Example

FIG. 7 illustrates another preferred binary masking scheme which isreferred to herein as the gray code masking scheme. According to thisembodiment, the masks m1 to m5 are selected such that a side of anygiven synthesis region is defined by the edge of only one mask. The siteat which the sequence BCDE is formed, for example, has its right edgedefined by m5 and its left side formed by mask m4 (and no other mask isaligned on the sides of this site). Accordingly, problems created bymisalignment, diffusion of light under the mask and the like will beminimized.

3. Example

FIG. 8 illustrates another binary masking scheme. According to thisscheme, referred to herein as a modified gray code masking scheme, thenumber of masks needed is minimized. For example, the mask m2 could bethe same mask as m1 and simply translated laterally. Similarly, the maskm4 could be the same as mask m3 and simply translated laterally.

4. Example

A four-step synthesis is shown in FIG. 9a. The reactants are the orderedset (A,B,C,D). In the first cycle, illumination through m₁ activates theupper half of the synthesis area. Building block A is then added to givethe distribution 602. Illumination through mask m₂ (which activates thelower half), followed by addition of B yields the next intermediatedistribution 604. C is added after illumination through m₃ (whichactivates the left half) giving the distribution 604, and D afterillumination through m₄ (which activates the right half), to yield thefinal product pattern 608 {AC,AD,BC,BD}.

5. Example

The above masking strategy for the synthesis may be extended for all 400dipeptides from the 20 naturally occurring amino acids as shown in FIG.9b. The synthesis consists of two rounds, with 20 photolysis andchemical coupling cycles per round. In the first cycle of round 1, mask1 activates 1/20th of the substrate for coupling with the first of 20amino acids. Nineteen subsequent illumination/coupling cycles in round 1yield a substrate consisting of 20 rectangular stripes each bearing adistinct member of the 20 amino acids. The masks of round 2 areperpendicular to round 1 masks and therefore a singleillumination/coupling cycle in round 2 yields 20 dipeptides. The 20illumination/coupling cycles of round 2 complete the synthesis of the400 dipeptides.

6. Example

The power of the binary masking strategy can be appreciated by theoutcome of a 10-step synthesis that produced 1,024 peptides. Thepolynomial expression for this 10-step binary synthesis was:

    (f+φ)(Y+φ)(G+φ)(A+φ)(G+φ)(T+φ)(F+φ)(L+φ)(S+φ)(F+φ)

Each peptide occupied a 400×400 μm square. A 32×32 peptide array (1,024peptides, including the null peptide and 10 peptides of l=1, and alimited number of duplicates) was clearly evident in a fluorescence scanfollowing side group deprotection and treatment with the antibody 3E7and fluorescinated antibody. Each synthesis site was a 400×400 μmsquare.

The scan showed a range of fluorescence intensities, from a backgroundvalue of 3,300 counts to 22,400 counts in the brightest square (x=20,y=9). Only 15 compounds exhibited an intensity greater than 12,300counts. The median value of the array was 4,800 counts.

The identity of each peptide in the array could be determined from its xand y coordinates (each range from 0 to 31) and the map of FIG. 10. Thechemical units at positions 2, 5, 6, 9, and 10 are specified by the ycoordinate and those at positions 1, 3, 4, 7, 8 by the x coordinate. Allbut one of the peptides was shorter than 10 residues. For example, thepeptide at x=12 and y=3 is YGAGF (SEQ ID NO:3) (positions 1, 6, 8, 9,and 10 are nulls). YGAFLS (SEQ ID NO:4), the brightest element of thearray, is at x=20 and y=9.

It is often desirable to deduce a binding affinity of a given peptidefrom the measured fluorescence intensity. Conceptually, the simplestcase is one in which a single peptide binds to a univalent antibodymolecule. The fluorescence scan is carried out after the slide is washedwith buffer for a defined time. The order of fluorescence intensities isthen a measure primarily of the relative dissociation rates of theantibody-peptide complexes. If the on-rate constants are the same (e.g.,if they are diffusion-controlled), the order of fluorescence intensitieswill correspond to the order of binding affinities. However, thesituation is sometimes more complex because a bivalent primary antibodyand a bivalent secondary antibody are used. The density of peptides in asynthesis area corresponded to a mean separation of ˜7 nm, which wouldallow multivalent antibody-peptide interactions. Hence, fluorescenceintensities obtained according to the method herein will often be aqualitative indicator of binding affinity.

Another important consideration is the fidelity of synthesis. Deletionsare produced by incomplete photodeprotection or incomplete coupling. Thecoupling yield per cycle in these experiments is typically between 85%and 95%. Implementing the switch matrix by masking is imperfect becauseof light diffraction, internal reflection, and scattering. Consequently,stowaways (chemical units that should not be on board) arise byunintended illumination of regions that should be dark. A binarysynthesis array contains many of the controls needed to assess thefidelity of a synthesis. For example, the fluorescence signal from asynthesis area nominally containing a tetrapeptide ABCD could come froma tripeptide deletion impurity such as ACD. Such an artifact would beruled out by the finding that the fluorescence intensity of the ACD siteis less than that of the ABCD site.

The fifteen most highly labelled peptides in the array obtained with thesynthesis of 1,024 peptides described above, were YGAFLS, YGAFS (SEQ IDNO:5), YGAFL (SEQ ID NO:6), YGGFLS (SEQ ID NO:7), YGAF (SEQ ID NO:8),YGALS (SEQ ID NO:9), YGGFS (SEQ ID NO:10), YGAL(SEQ ID NO:11), YGAFLF(SEQ ID NO:12), YGAF, YGAFF (SEQ ID NO:13), YGGLS (SEQ ID NO:14), YGGFL(SEQ ID NO:15), YGAFSF (SEQ ID NO:16), and YGAFLSF (SEQ ID NO:17). Astriking feature is that all fifteen begin with YG, which agrees withprevious work showing that an amino-terminal tyrosine is a keydeterminant of binding. Residue 3 of this set is either A or G, andresidue 4 is either F or L. The exclusion of S and T from thesepositions is clear cut. The finding that the preferred sequence is YG(A/G) (F/L) fits nicely with the outcome of a study in which a verylarge library of peptides on phage generated by recombinant DNA methodswas screened for binding to antibody 3E7 (see Cwirla et al., Proc. Natl.Acad. Sci. USA, (1990) 87:6378, incorporated herein by reference).Additional binary syntheses based on leads from peptides on phageexperiments show that YGAFMQ (SEQ ID NO:18), YGAFM (SEQ ID NO:19), andYGAFQ (SEQ ID NO:20) give stronger fluorescence signals than does YGGFM,the immunogen used to obtain antibody 3E7.

Variations on the above masking strategy will be valuable in certaincircumstances. For example, if a "kernel" sequence of interest consistsof PQR separated from XYZ and the aim is to synthesize peptides in whichthese units are separated by a variable number of different residues,then the kernel can be placed in each peptide by using a mask that has1's everywhere. The polynomial representation of a suitable synthesisis:

    (P)(Q)(R)(A+φ)(B+φ)(C+φ)(D+φ)(X)(Y)(Z)

Sixteen peptides will be formed, ranging in length from the 6-mer PQRXYZto the 10-mer PQRABCDXYZ.

Several other masking strategies will also find value in selectedcircumstances. By using a particular mask more than once, two or morereactants will appear in the same set of products. For example, supposethat the mask for an 8-step synthesis is

A 11110000

B 00001111

C 11001100

D 00110011

E 10101010

F 01010101

G 11110000

H 00001111

The products are ACEG, ACFG, ADEG, ADFG, BCEH, BCFH, BDEH, and BDFH. Aand G always appear together because their additions were directed bythe same mask, and likewise for B and H.

C. Linker Selection

According to preferred embodiments the linker molecules used as anintermediary between the synthesized polymers and the substrate areselected for optimum length and/or type for improved binding interactionwith a receptor. According to this aspect of the invention diverselinkers of varying length and/or type are synthesized for subsequentattachment of a ligand. Through variations in the length and type oflinker, it becomes possible to optimize the binding interaction betweenan immobilized ligand and its receptor.

The degree of binding between a ligand (peptide, inhibitor, hapten,drug, etc.) and its receptor (enzyme, antibody, etc.) when one of thepartners is immobilized on to a substrate will in some embodimentsdepend on the accessibility of the receptor in solution to theimmobilized ligand. The accessibility in turn will depend on the lengthand/or type of linker molecule employed to immobilize one of thepartners. Preferred embodiments of the invention therefore employ theVLSIPS™ technology described herein to generate an array of, preferably,inactive or inert linkers of varying length and/or type, usingphotochemical protecting groups to selectively expose different regionsof the substrate and to build upon chemically-active group.

In the simplest embodiment of this concept, the same unit is attached tothe substrate in varying multiples or lengths in known locations on thesubstrate via VLSIPS techniques to generate an array of polymers ofvarying length. A single ligand (peptide, drug, hapten, etc.) isattached to each of them, and an assay is performed with the bindingsite to evaluate the degree of binding with a receptor that is known tobind to the ligand. In cases where the linker length impacts the abilityof the receptor to bind to the ligand, varying levels of binding will beobserved. In general, the linker which provides the highest binding willthen be used to assay other ligands synthesized in accordance with thetechniques herein.

According to other embodiments the binding between a singleligand/receptor pair is evaluated for linkers of diverse monomersequence. According to these embodiments, the linkers are synthesized inan array in accordance with the techniques herein and have differentmonomer sequence (and, optionally, different lengths). Thereafter, allof the linker molecules are provided with a ligand known to have atleast some binding affinity for a given receptor. The given receptor isthen exposed to the ligand and binding affinity is deduced. Linkermolecules which provide adequate binding between the ligand and receptorare then utilized in screening studies.

D. Protecting Groups

As discussed above, selectively removable protecting groups allowcreation of well defined areas of substrate surface having differingreactivities. Preferably, the protecting groups are selectively removedfrom the surface by applying a specific activator, such aselectromagnetic radiation of a specific wavelength and intensity. Morepreferably, the specific activator exposes selected areas of surface toremove the protecting groups in the exposed areas.

Protecting groups of the present invention are used in conjunction withsolid phase oligomer syntheses, such as peptide syntheses using naturalor unnatural amino acids, nucleotide syntheses using deoxyribonucleicand ribonucleic acids, oligosaccharide syntheses, and the like. Inaddition to protecting the substrate surface from unwanted reaction, theprotecting groups block a reactive end of the monomer to preventself-polymerization. For instance, attachment of a protecting group tothe amino terminus of an activated amino acid, such as anN-hydroxysuccinimide-activated ester of the amino acid, prevents theamino terminus of one monomer from reacting with the activated esterportion of another during peptide synthesis. Alternatively, theprotecting group may be attached to the carboxyl group of an amino acidto prevent reaction at this site. Most protecting groups can be attachedto either the amino or the carboxyl group of an amino acid, and thenature of the chemical synthesis will dictate which reactive group willrequire a protecting group. Analogously, attachment of a protectinggroup to the 5'-hydroxyl group of a nucleoside during synthesis usingfor example, phosphate-triester coupling chemistry, prevents the5'-hydroxyl of one nucleoside from reacting with the 3'-activatedphosphate-triester of another.

Regardless of the specific use, protecting groups are employed toprotect a moiety on a molecule from reacting with another reagent.Protecting groups of the present invention have the followingcharacteristics: they prevent selected reagents from modifying the groupto which they are attached; they are stable (that is, they remainattached to the molecule) to the synthesis reaction conditions; they areremovable under conditions that do not adversely affect the remainingstructure; and once removed, do not react appreciably with the surfaceor surface-bound oligomer. The selection of a suitable protecting groupwill depend, of course, on the chemical nature of the monomer unit andoligomer, as well as the specific reagents they are to protect against.

In a preferred embodiment, the protecting groups are photoactivatable.The properties and uses of photoreactive protecting compounds have beenreviewed. See, McCray et al., Ann. Rev. of Biophys. and Biophys. Chem.(1989) 18:239-270, which is incorporated herein by reference.Preferably, the photosensitive protecting groups will be removable byradiation in the ultraviolet (UV) or visible portion of theelectromagnetic spectrum. More preferably, the protecting groups; willbe removable by radiation in the near UV or visible portion of thespectrum. In some embodiments, however, activation may be performed byother methods such as localized heating, electron beam lithography,laser pumping, oxidation or reduction with microelectrodes, and thelike. Sulfonyl compounds are suitable reactive groups; for electron beamlithography. Oxidative or reductive removal is accomplished by exposureof the protecting group to an electric current source, preferably usingmicroelectrodes directed to the predefined regions of the surface whichare desired for activation. Other methods may be used in light of thisdisclosure.

Many, although not all, of the photoremovable protecting groups will bearomatic compounds that absorb near-UV and visible radiation. Suitablephotoremovable protecting groups are described in, for example, McCrayet al., Patchornik, J. Amer. Chem. Soc. (1970) 92:6333, and Amit et al.,J. Org. Chem. (1974) 39:192, which are incorporated herein by reference.

A preferred class of photoremovable protecting groups has the generalformula: ##STR3## where R¹, R², R³, and R⁴ independently are a hydrogenatom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol,thioether, amino, nitro, carbokyl, formate, formamido or phosphidogroup, or adjacent substituents (i.e., R¹ -R², R² -R³, R³ -R⁴) aresubstituted oxygen groups that together form a cyclic acetal or ketal;R⁵ is a hydrogen atom, a alkoxyl, alkyl, hydrogen, halo, aryl, oralkenyl group, and n=0 or 1.

A preferred protecting group, 6-nitroveratryl (NV), which is used forprotecting the carboxyl terminus of an amino acid or the hydroxyl groupof a nucleotide, for example, is formed when R² and R³ are each amethoxy group, R¹, R⁴ and R⁵ are each a hydrogen atom, and n=0: ##STR4##

A preferred protecting group, 6-nitroveratryloxycarbonyl (NVOC), whichis used to protect the amino terminus of an amino acid, for example, isformed when R² and R³ are each a methoxy group, R¹, R⁴ and R⁵ are each ahydrogen atom, and n=1: ##STR5##

Another preferred protecting group, 6-nitropiperonyl (NP), which is usedfor protecting the carboxyl terminus of an amino acid or the hydroxylgroup of a nucleotide, for example, is formed when R² and R³ togetherform a methylene acetal, R¹, R⁴ and R⁵ are each a hydrogen atom, andn=0: ##STR6##

Another preferred protecting group, 6-nitropiperonyloxycarbonyl (NPOC),which is used to protect the amino terminus of an amino acid, forexample, is formed when R² and R³ together form a methylene acetal, R¹,R⁴ and R⁵ are each a hydrogen atom, and n=1: ##STR7##

A most preferred protecting group, methyl-6-nitroveratryl (MeNV), whichis used for protecting the carboxyl terminus of an amino acid or thehydroxyl group of a nucleotide, for example, is formed when R² and R³are each a methoxy group, R¹ and R⁴ are each a hydrogen atom, R⁵ is amethyl group, and n=0: ##STR8##

Another most preferred protecting group,methyl-6-nitroveratryloxycarbonyl (MeNVOC), which is used to protect theamino terminus of an amino acid, for example, is formed when R² and R³are each a methoxy group, R¹ and R⁴ are each a hydrogen atom, R⁵ is amethyl group, and n=1: ##STR9##

Another most preferred protecting group, methyl-6-nitropiperonyl (MeNP),which is used for protecting the carboxyl terminus of an amino acid orthe hydroxyl group of a nucleotide, for example, is formed when R² andR³ together form a methylene acetal, R¹ and R⁴ are each a hydrogen atom,R⁵ is a methyl group, and n=0: ##STR10##

Another most preferred protecting group,methyl-6-nitropiperonyloxycarbonyl (MeNPOC), which is used to protectthe amino terminus of an amino acid, for example, is formed when R² andR³ together form a methylene acetal, R¹ and R⁴ are each a hydrogen atom,R⁵ is a methyl group, and n=1: ##STR11##

A protected amino acid having a photoactivatable oxycarbonyl protectinggroup, such NVOC or NPOC or their corresponding methyl derivatives,MeNVOC or MeNPOC, respectively, on the amino terminus is formed byacylating the amine of the amino acid with an activated oxycarbonylester of the protecting group. Examples of activated oxycarbonyl estersof NVOC and MeNVOC have the general formula: ##STR12## where X ishalogen, mixed anhydride, phenoxy, p-nitrophenoxy, N-hydroxysuccinimide,and the like.

A protected amino acid or nucleotide having a photoactivatableprotecting group, such as NV or NP or their corresponding methylderivatives, MeNV or MeNP, respectively, on the carboxy terminus of theamino acid or 5'-hydroxy terminus of the nucleotide, is formed byacylating the carboxy terminus or 5'-OH with an activated benzylderivative of the protecting group. Examples of activated benzylderivatives of MeNV and MeNP have the general formula: ##STR13## where Xis halogen, hydroxyl, tosyl, mesyl, trifluormethyl, diazo, azido, andthe like.

Another method for generating protected monomers is to react thebenzylic alcohol derivative of the protecting group with an activatedester of the monomer. For example, to protect the carboxyl terminus ofan amino acid, an activated ester of the amino acid is reacted with thealcohol derivative of the protecting group, such as 6-nitroveratrol(NVOH). Examples of activated esters suitable for such uses includehalo-formate, mixed anhydride, imidazoyl formate, acyl halide, and alsoincludes formation of the activated ester in situ the use of commonreagents such as DCC and the like. See Atherton et al. for otherexamples of activated esters.

A further method for generating protected monomers is to react thebenzylic alcohol derivative of the protecting group with an activatedcarbon of the monomer. For example, to protect the 5'-hydroxyl group ofa nucleic acid, a derivative having a 5'-activated carbon is reactedwith the alcohol derivative of the protecting group, such asmethyl-6-nitropiperonol (MePyROH). Examples of nucleotides havingactivating groups attached to the 5'-hydroxyl group have the generalformula: ##STR14## where Y is a halogen atom, a tosyl, mesyl,trifluoromethyl, azido, or diazo group, and the like.

Another class of preferred photochemical protecting groups has theformula: ##STR15## where R¹, R², and R³ independently are a hydrogenatom, a lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol,thioether, amino, nitro, carboxyl, formate, formamido, sulfanates,sulfido or phosphido group, R⁴ and R⁵ independently are a hydrogen atom,an alkoxy, alkyl, halo, aryl, hydrogen, or alkenyl group, and n=0 or 1.

A preferred protecting group, 1-pyrenylmethyloxycarbonyl (PyROC), whichis used to protect the amino terminus of an amino acid, for example, isformed when R¹ through R⁵ are each a hydrogen atom and n=1: ##STR16##

Another preferred protecting group, 1-pyrenylmethyl (PyR), which is usedfor protecting the carboxy terminus of an amino acid or the hydroxylgroup of a nucleotide, for example, is formed when R¹ through R⁵ areeach a hydrogen atom and n=0: ##STR17##

An amino acid having a pyrenylmethyloxycarbonyl protecting group on itsamino terminus is formed by acylation of the free amine of amino acidwith an activated oxycarbonyl ester of the pyrenyl protecting group.Examples of activated oxycarbonyl esters of PyROC have the generalformula: ##STR18## where X is halogen, or mixed anhydride,p-nitrophenoxy, or N-hydroxysuccinimide group, and the like.

A protect ed amino acid or nucleotide having a photoactivatableprotecting group, such as PyR, on the carboxy terminus of the amino acidor 5'-hydroxy terminus of the nucleic acid, respectively, is formed byacylating the carboxy terminus or 5'-OH with an activated pyrenylmethylderivative of the protecting group. Examples of activated pyrenylmethylderivatives of PyR have the general formula: ##STR19## where X is ahalogen atom, a hydroxyl, diazo, or azido group, and the like.

Another method of generating protected monomers is to react thepyrenylmethyl alcohol moiety of the protecting group with an activatedester of the monomer. For example, an activated ester of an amino acidcan be reacted with the alcohol derivative of the protecting group, suchas pyrenylmethyl alcohol (PyROH), to form the protected derivative ofthe carboxy terminus of the amino acid. Examples of activated estersinclude halo-formate, mixed anhydride, imidazoyl formate, acyl halide,and also includes formation of the activated ester in situ and the useof common reagents such as DCC and the like.

Clearly, many photosensitive protecting groups are suitable for use inthe present invention.

In preferred embodiments, the substrate is irradiated to remove thephotoremovable protecting groups and create regions having free reactivemoieties and side products resulting from the protecting group. Theremoval rate of the protecting groups depends on the wavelength andintensity of the incident radiation, as well as the physical andchemical properties of the protecting group itself. Preferred protectinggroups are removed at a faster rate and with a lower intensity ofradiation. For example, at a given set of conditions, MeNVOC and MeNPOCare photolytically removed from the N-terminus of a peptide chain fasterthan their unsubstituted parent compounds, NVOC and NPOC, respectively.

Removal of the protecting group is accomplished by irradiation toliberate the reactive group and degradation products derived from theprotecting group. Not wishing to be bound by theory, it is believed thatirradiation of an NVOC- and MeNVOC-protected oligomers occurs by thefollowing reaction schemes:

    NVOC-AA→3,4-dimethoxy-6-nitrosobenzaldehyde+Co.sub.2 +AA

    MeNVOC-AA→3,4-dimethoxy-6-nitrosoacetophenone+Co.sub.2 +AA

where AA represents the N-terminus of the amino acid oligomer.

Along with the unprotected amino acid, other products are liberated intosolution: carbon dioxide and a 2,3-dimethoxy-6-nitrosophenylcarbonylcompound, which can react with nucleophilic portions of the oligomer toform unwanted secondary reactions. In the case of an NVOC-protectedamino acid, the degradation product is a nitrosobenzaldehyde, while thedegradation product for the other is a nitrosophenyl ketone. Forinstance, it is believed that the product aldehyde from NVOC degradationreacts with free amines to form a Schiff base (imine) that affects theremaining polymer synthesis. Preferred photoremovable protecting groupsreact slowly or reversibly with the oligomer on the support.

Again not wishing to be bound by theory, it is believed that the productketone from irradiation of a MeNVOC-protected oligomer reacts at aslower rate with nucleophiles on the oligomer than the product aldehydefrom irradiation of the same NVOC-protected oligomer. Although notunambiguously determined, it is believed that this difference inreaction rate is due to the difference in general reactivity betweenaldehyde and ketones towards nucleophiles due to steric and electroniceffects.

The photoremovable protecting groups of the present invention arereadily removed. For example, the photolysis of N-protectedL-phenylalanine in solution and having different photoremovableprotecting groups was analyzed, and the results are presented in thefollowing table:

                  TABLE                                                           ______________________________________                                        Photolysis of Protected L--Phe--OH                                                        T.sub.1/2  in seconds                                             Solvent       NBOC    NVOC    MeNVOC MeNPOC                                   ______________________________________                                        Dioxane       1288    110     24     19                                       5 mM H.sub.2 SO.sub.4 /Dioxane                                                              1575    98      33 22                                           ______________________________________                                    

The half life, t_(1/2), is the time in seconds required to remove 50% ofthe starting amount of protecting group. NBOC is the6-nitrobenzyloxycarbonyl group, NVOC is the 6-nitroveratryloxycarbonylgroup, MeNVOC is the methyl-6-nitroveratryloxycarbonyl group, and MeNPOCis the methyl-6-nitropiperonyloxycarbonyl group. The photolysis wascarried out in the indicated solvent with 362/364 nm-wavelengthirradiation having an intensity of 10 mW/cm², and the concentration ofeach protected phenylalanine was 0.10 mM.

The table shows that deprotection of NVOC-, MeNVOC-, andMeNPOC-protected phenylalanine proceeded faster than the deprotection ofNBOC. Furthermore, it shows that the deprotection of the two derivativesthat are substituted on the benzylic carbon, MeNVOC and MeNPOC, werephotolyzed at the highest rates in both dioxane and acidified dioxane.

1. Use of Photoremovable Groups During Solid-Phase Synthesis of Peptides

The formation of peptides on a solid-phase support requires the stepwiseattachment of an amino acid to a substrate-bound growing chain. In orderto prevent unwanted polymerization of the monomeric amino acid under thereaction conditions, protection of the amino terminus of the amino acidis required. After the monomer is coupled to the end of the peptide, theN-terminal protecting group is removed, and another amino acid iscoupled to the chain. This cycle of coupling and deprotecting iscontinued for each amino acid in the peptide sequence. See Merrifield,J. Am. Chem. Soc. (1963) 85:2149, and Atherton et al., "Solid PhasePeptide Synthesis" 1989, IRL Press, London, both incorporated herein byreference for all purposes. As described above, the use of aphotoremovable protecting group allows removal of selected portions ofthe substrate surface, via patterned irradiation, during thedeprotection cycle of the solid phase synthesis. This selectively allowsspatial control of the synthesis--the next amino acid is coupled only tothe irradiated areas.

In one embodiment, the photoremovable protecting groups of the presentinvention are attached to an activated ester of an amino acid at theamino terminus: ##STR20## where R is the side chain of a natural orunnatural amino acid, X is a photoremovable protecting group, and Y isan activated carboxylic acid derivative. The photoremovable protectinggroup, X, is preferably NVOC, NPOC, PyROC, MeNVOC, MeNPOC, and the likeas discussed above. The activated ester, Y, is preferably a reactivederivative having a high coupling efficiency, such as an acyl halide,mixed anhydride, N-hydroxysuccinimide ester, perfluorophenyl ester, orurethane protected acid, and the like. Other activated esters andreaction conditions are well known (See Atherton et al.).

2. Use of Photoremovable Groups During Solid-Phase Synthesis ofOligonucleotides

The formation of oligonucleotides on a solid-phase support requires thestepwise attachment of a nucleotide to a substrate-bound growingoligomer. In order to prevent unwanted polymerization of the monomericnucleotide under the reaction conditions, protection of the 5'-hydroxylgroup of the nucleotide is required. After the monomer is coupled to theend of the oligomer, the 5'-hydroxyl protecting group is removed, andanother nucleotide is coupled to the chain. This cycle of coupling anddeprotecting is continued for each nucleotide in the oligomer sequence.See Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRLPress, London, incorporated herein by reference for all purposes. Asdescribed above, the use of a photoremovable protecting group allowsremoval, via patterned irradiation, of selected portions of thesubstrate surface during the deprotection cycle of the solid phasesynthesis. This selectively allows spatial control of the synthesis--thenext nucleotide is coupled only to the irradiated areas.

Oligonucleotide synthesis generally involves coupling an activatedphosphorous derivative on the 3'-hydroxyl group of a nucleotide with the5'-hydroxyl group of an oligomer bound to a solid support. Two majorchemical methods exist to perform this coupling: the phosphate-triesterand phosphoramidite methods (See Gait). Protecting groups of the presentinvention are suitable for use in either method.

In a preferred embodiment, a photoremovable protecting group is attachedto an activated nucleotide on the 5'-hydroxyl group: ##STR21## where Bis the base attached to the sugar ring; R is a hydrogen atom when thesugar is deoxyribose or R is a hydroxyl group when the sugar is ribose;P represents an activated phosphorous group; and X is a photoremovableprotecting group. The photoremovable protecting group, X, is preferablyNV, NP, PyR, MeNV, MeNP, and the like as described above. The activatedphosphorous group, P, is preferably a reactive derivative having a highcoupling efficiency, such as a phosphate-triester, phosphoramidite orthe like. Other activated phosphorous derivatives, as well as reactionconditions, are well known (See Gait).

E. Amino Acid N-Carboxy Anhydrides Protected With a Photoremovable Group

During Merrifield peptide synthesis, an activated ester of one aminoacid is coupled with the free amino terminus of a substrate-boundoligomer. Activated esters of amino acids suitable for the solid phasesynthesis include halo-formate, mixed anhydride, imidazoyl formate, acylhalide, and also includes formation of the activated ester in situ andthe use of common reagents such as DCC and the like (See Atherton etal.). A preferred protected and activated amino acid has the generalformula: ##STR22## where R is the side chain of the amino acid and X isa photoremovable protecting group. This compound is a urethane-protectedamino acid having a photoremovable protecting group attach to the amine.A more preferred activated amino acid is formed when the photoremovableprotecting group has the general formula: ##STR23## where R¹, R², R³,and R⁴ independently are a hydrogen atom, a lower alkyl, aryl, benzyl,halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl,formate, formamido or phosphido group, or adjacent substituents (i.e.,R¹ -R², R² -R³, R³ -R⁴) are substituted oxygen groups that together forma cyclic acetal or ketal; and R⁵ is a hydrogen atom, an alkoxyl, alkyl,hydrogen, halo, aryl, or alkenyl group.

A preferred activated amino acid is formed when the photoremovableprotecting group is 6-nitroveratryloxycarbonyl. That is, R¹ and R⁴ areeach a hydrogen atom, R² and R³ are each a methoxy group, and R⁵ is ahydrogen atom. Another preferred activated amino acid is formed when thephotoremovable group is 6-nitropiperonyl: R¹ and R⁴ are each a hydrogenatom, R² and R³ together form a methylene acetal, and R⁵ is a hydrogenatom. Other protecting groups are possible. Another preferred activatedester is formed when the photoremovable group is methyl-6-nitroveratrylor methyl-6-nitropiperonyl.

Another preferred activated amino acid is formed when the photoremovableprotecting group has the general formula: ##STR24## where R¹, R², and R³independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen,hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate,formamido, sulfanates, sulfido or phosphido group, and R⁴ and R⁵independently are a hydrogen atom, an alkoxy, alkyl, halo, aryl,hydrogen, or alkenyl group. The resulting compound is aurethane-protected amino acid having a pyrenylmethyloxycarbonylprotecting group attached to the amine. A more preferred embodiment isformed when R¹ through R⁵ are each a hydrogen atom.

The urethane-protected amino acids having a photoremovable protectinggroup of the present invention are prepared by condensation of anN-protected amino acid with an acylating agent such as an acyl halide,anhydride, chloroformate and the like (See Fuller et al., U.S. Pat. No.4,946,942 and Fuller et al., J. Amer. Chem. Soc. (1990) 112:7414-7416,both herein incorporated by reference for all purposes).

Urethane-protected amino acids having photoremovable protecting groupsare generally useful as reagents during solid-phase peptide synthesis,and because of the spatially selectivity possible with thephotoremovable protecting group, are especially useful for the spatiallyaddressable peptide synthesis. These amino acids are difunctional: theurethane group first serves to activate the carboxy terminus forreaction with the amine bound to the surface and, once the peptide bondis formed, the photoremovable protecting group protects the newly formedamino terminus from further reaction. These amino acids are also highlyreactive to nucleophiles, such as deprotected amines on the surface ofthe solid support, and due to this high reactivity, the solid-phasepeptide coupling times are significantly reduced, and yields aretypically higher.

IV. Data Collection

A. Data Collection System

Substrates prepared in accordance with the above description are used inone embodiment to determine which of the plurality of sequences thereonbind to a receptor of interest. FIG. 11 illustrates one embodiment of adevice used to detect regions of a substrate which contain fluorescentmarkers. This device would be used, for example, to detect the presenceor absence of a labeled receptor such as an antibody which has bound toa synthesized polymer on a substrate.

Light is directed at the substrate from a light source 1002 such as alaser light source of the type well known to those of skill in the artsuch as a model no. 2025 made by Spectra Physics. Light from the sourceis directed at a lens 1004 which is preferably a cylindrical lens of thetype well known to those of skill in the art. The resulting output fromthe lens 1004 is a linear beam rather than a spot of light, resulting inthe capability to detect data substantially simultaneously along alinear array of pixels rather than on a pixel-by-pixel basis. It will beunderstood that a cylindrical lens is used herein as an illustration ofone technique for generating a linear beam of light on a surface, butthat other techniques could also be utilized.

The beam from the cylindrical lens is passed through a dichroic mirroror prism (1006) and directed at the surface of the suitably preparedsubstrate 1008. Substrate 1008 is placed on an x-y translation stage1009 such as a model no. PM500-8 made by Newport. Light at certainlocations on the substrate will be fluoresced and transmitted along thepath indicated by dashed lines back through the dichroic mirror, andfocused with a suitable lens 1010 such as an f/1.4 camera lens on alinear detector 1012 via a variable f stop focusing lens 1014. Throughuse of a linear light beam, it becomes possible to generate data over aline of pixels (such as about 1 cm) along the substrate, rather thanfrom individual points on the substrate. In alternative embodiments,light is directed at a 2-dimensional area of the substrate andfluoresced light detected by a 2-dimensional CCD array. Linear detectionis preferred because substantially higher power densities are obtained.

Detector 1012 detects the amount of light fluoresced from the substrateas a function of position. According to one embodiment the detector is alinear CCD array of the type commonly known to those of skill in theart. The x-y translation stage, the light source, and the detector 1012are all operably connected to a computer 1016 such as an IBM PC-AT orequivalent for control of the device and data collection from the CCDarray.

In operation, the substrate is appropriately positioned by thetranslation stage. The light source is then illuminated, and intensitydata are gathered with the computer via the detector.

FIG. 12 illustrates the architecture of the data collection system ingreater detail. Operation of the system occurs under the direction ofthe photon counting program 1102 (photon), included herewith as AppendixB. The user inputs the scan dimensions, the number of pixels or datapoints in a region, and the scan speed to the counting program. Via aGP1B bus 1104 the program (in an IBM PC compatible computer, forexample) interfaces with a multichannel scaler 1106 such as a StanfordResearch SR 430 and an x-y stage controller 1108 such as a PM500. Thesignal from the light from the fluorescing substrate enters a photoncounter 1110, providing output to the scaler 1106. Data are output fromthe scaler indicative of the number of counts in a given region. Afterscanning a selected area, the stage controller is activated withcommands for acceleration and velocity, which in turn drives the scanstage 1112 such as a PM500-A to another region.

Data are collected in an image data file 1114 and processed in a scalingprogram 1116, also included in Appendix B. A scaled image is output fordisplay on, for example, a VGA display 1118. The image is scaled basedon an input of the percentage of pixels to clip and the minimum andmaximum pixel levels to be viewed. The system outputs for use the minand max pixel levels in the raw data.

B. Data Analysis

The output from the data collection system is an array of dataindicative of fluorescent intensity versus location on the substrate.The data are typically taken over regions substantially smaller than thearea in which synthesis of a given polymer has taken place. Merely byway of example, if polymers were synthesized in squares on the substratehaving dimensions of 500 microns by 500 microns, the data may be takenover regions having dimensions of 5 microns by 5 microns. In mostpreferred embodiments, the regions over which florescence data are takenacross the substrate are less than about 1/2 the area of the regions inwhich individual polymers are synthesized, preferably less than 1/10 thearea in which a single polymer is synthesized, and most preferably lessthan 1/100 the area in which a single polymer is synthesized. Hence,within any area in which a given polymer has been synthesized, a largenumber of fluorescence data points are collected.

A plot of number of pixels versus intensity for a scan of a cell when ithas been exposed to, for example, a labeled antibody will typically takethe form of a bell curve, but spurious data are observed, particularlyat higher intensities. Since it is desirable to use an average offluorescent intensity over a given synthesis region in determiningrelative binding affinity, these spurious data will tend to undesirablyskew the data.

Accordingly, in one embodiment of the invention the data are correctedfor removal of these spurious data points, and an average of the datapoints is thereafter utilized in determining relative bindingefficiency.

FIG. 13 illustrates one embodiment of a system for removal of spuriousdata from a set of fluorescence data such as data used in affinityscreening studies. A user or the system inputs data relating to the chiplocation and cell corners at step 1302. From this information and theimage file, the system creates a computer representation of a histogramat step 1304, the histogram (at least in the form of a computer file)plotting number of data pixels versus intensity.

For each cell, a main data analysis loop is then performed. For eachcell, at step 1306, the system calculates the total intensity or numberof pixels for the bandwidth centered around varying intensity levels.For example, as shown in the plot to the right of step 1306, the systemcalculates the number of pixels within the band of width w. The systemthen "moves" this bandwidth to a higher center intensity, and againcalculates the number of pixels in the bandwidth. This process isrepeated until the entire range of intensities has been scanned, and atstep 1308 the system determines which band has the highest total numberof pixels. The data within this bandwidth are used for further analysis.Assuming the bandwidth is selected to be reasonably small, thisprocedure will have the effect of eliminating spurious data located atthe higher intensity levels. The system then repeats at step 1310 if allcells have been evaluated, or repeats for the next cell.

At step 1312 the system then integrates the data within the bandwidthfor each of the selected cells, sorts the data at step 1314 using thesynthesis procedure file, and displays the data to a user on, forexample, a video display or a printer.

V. Representative Applications

A. Oligonucleotide Synthesis

The generality of light directed spatially addressable parallel chemicalsynthesis is demonstrated by application to nucleic acid synthesis.

1. Example

Light activated formation of a thymidine-cytidine dimer was carried out.A three dimensional representation of a fluorescence scan showing acheckerboard pattern generated by the light-directed synthesis of adinucleotide is shown in FIG. 8. 5'-nitroveratryl thymidine was attachedto a synthesis substrate through the 3' hydroxyl group. Thenitroveratryl protecting groups were removed by illumination through a500 mm checkerboard mask. The substrate was then treated withphosphoramidite activated 2'-deoxycytidine. In order to follow thereaction fluorometrically, the deoxycytidine had been modified with anFMOC protected aminohexyl linker attached to the exocyclic amine(5'-O-dimethoxytrityl-4-N-(6-N-fluorenylmethylcarbamoyl-hexylcarboxy)-2'-deoxycytidine).After removal of the FMOC protecting group with base, the regions whichcontained the dinucleotide were fluorescently labelled by treatment ofthe substrate with 1 mM FITC in DMF for one hour.

The three-dimensional representation of the fluorescent intensity datain FIG. 14 clearly reproduces the checkerboard illumination pattern usedduring photolysis of the substrate. This result demonstrates thatoligonucleotides as well as peptides can be synthesized by thelight-directed method.

VI. Conclusion

The inventions herein provide a new approach for the simultaneoussynthesis of a large number of compounds. The method can be appliedwhenever one has chemical building blocks that can be coupled in asolid-phase format, and when light can be used to generate a reactivegroup.

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example, whilethe invention is illustrated primarily with regard to peptide andnucleotide synthesis, the invention is not so limited. The scope of theinvention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 21                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TyrGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TyrGlyAlaGlyPhe                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TyrGlyAlaPheLeuSer                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TyrGlyAlaPheSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TyrGlyAlaPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TyrGlyGlyPheLeuSer                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       TyrGlyAlaPhe                                                                  (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TyrGlyAlaLeuSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TyrGlyGlyPheSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TyrGlyAlaLeu                                                                  1                                                                             (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      TyrGlyAlaPheLeuPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      TyrGlyAlaPhePhe                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TyrGlyGlyLeuSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TyrGlyGlyPheLeu                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TyrGlyAlaPheSerPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TyrGlyAlaPheLeuSerPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TyrGlyAlaPheMetGln                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TyrGlyAlaPheMet                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TyrGlyAlaPheGln                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      TyrGlyGlyPheMet                                                               15                                                                            __________________________________________________________________________

What is claimed is:
 1. A reactor system for synthesizing a plurality ofpolymer sequences on a substrate comprising:a flow cell; a substratemounted on said flow cell forming a cavity between said substrate andsaid flow cell; a system for delivering selected monomer containingreaction fluids to said cavity; a mask; a translation stage for movingsaid mask or said substrate from a first relative location to a secondrelative location; an energy source for directing energy through saidmask, at selected regions of said substrate at selected times; and anappropriately programmed digital computer for selectively directing aflow of reaction fluids into said cavity, selectively activating saidtranslation stage and selectively illuminating said substrate so as toform a plurality of diverse, known polymer sequences on said substrateat predetermined locations.
 2. The reactor system of claim 1, whereinsaid substrate has a first surface having reactive functional groupsthereon, said reactive functional groups being protected by a protectivegroup, said protective group being removable upon exposure to an energysource, said energy source being selected from the group consisting ofelectron beam radiation, gamma radiation, x-ray radiation, ultra-violetradiation, visible light, and infrared radiation.
 3. The reactor systemof claim 2, wherein said flow cell comprises a body having a cavitydisposed therein, said cavity being in fluid contact with said firstsurface of said substrate, and said cavity including at least an inletfor flowing monomer containing reaction fluids into said cavity.
 4. Thereactor system of claim 3, wherein said cavity comprises an outlet forflowing said solution containing monomers out of said cavity.
 5. Thereactor system of claim 3, wherein said substrate is mounted over saidcavity, thereby sealably covering said cavity whereby said first surfaceof said substrate is in fluid communication with said cavity.
 6. Thereactor system of claim 2, wherein said protective group is coupled to alinker group on said surface.
 7. The reactor system of claim 2, whereinsaid protective group is selected from the group consisting oforthonitrobenzyl derivatives, methyl-6-nitroveratryl,6-nitroveratryloxycarbonyl, methyl-6-nitroveratryloxycarbonyl,methyl-6-nitropiperonyl, 6-nitropiperonyloxycarbonyl,methyl-6-nitropiperonyloxycarbonyl, 2-nitrobenzyloxycarbonyl, andmixtures thereof.
 8. The reactor system of claim 1, wherein saidsubstrate comprises glass.
 9. The reactor system of claim 1, whereinsaid said mask comprises transparent locations and opaque locations. 10.The reactor system of claim 9, wherein said mask compriseschrome-on-glass.
 11. The apparatus of claim 1, wherein said polymersequences comprise oligonucleotide sequences and said system fordelivering selected monomer containing reaction fluids to said reactoris a system for delivering selected nucleic acid monomers to saidsurface for binding to said surface.